1. Field of the Invention
The present invention relates generally to conjugate molecules and more specifically, to conjugate molecules and their use in the delivery of biologically active agents into cells.
2. Background Information
Various delivery vehicles have been used to deliver biologically active molecules (e.g., peptides) into cells for therapeutic or diagnostic purposes. Several proteins and small peptides have the ability to transduce or travel through biological membranes independent of classical receptor- or endocytosis-mediated pathways. Examples of these proteins include the HIV-1 TAT protein, the herpes simplex virus 1 (HSV-1) DNA-binding protein VP22, and the third alpha helix of Drosophila Antennapedia (Ante) homeotic transcription factor. The small protein transduction domains (PTDs) from these proteins can be incorporated into peptide conjugates including a biologically active molecule to transport the conjugate into a cell. The potential disadvantage of these vectors is that they are foreign proteins that may be immunogenic in humans.
Certain anti-DNA autoantibodies have also been shown to penetrate cells and localize to the cell nucleus. Cellular penetration by anti-DNA antibodies was initially demonstrated in peripheral blood T-lymphocytes and, subsequently, was shown to affect their function. This phenomenon has allowed the use of certain antibodies in the generation of peptide conjugates capable of transporting a wide variety of biologically active materials, e.g., nuclear transcription factors, enzymes, enzyme inhibitors, genes, and the like, to the cell nucleus for a variety of therapeutic effects. Any variety of agents may be transported via conjugation to the antibody, or fragment of the antibody, such as inorganic and organic molecules, pharmaceutical agents, drugs, peptides, proteins, genetic material, and the like.
A particular class of monoclonal antibodies that is known to be utilized to transport a wide variety of biologically important molecules into target cells, such as kidney cells, brain cells, ovarian cells, bone cells, and the like is mAb 3E10 and mutants and/or functional fragments thereof. Monoclonal antibody 3E10 is produced by a hybridoma 3E10 placed permanently on deposit with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA, on Aug. 31, 2000, according to the terms of the Budapest Treaty under ATCC accession number PTA-2439. mAb 3E10 (or functional fragments thereof) can be conjugated to the biological molecule of interest to form an antibody conjugate that is capable of being transported into the cell. Upon entry into the cell, the antibody conjugate localizes in and around the cell nucleus. Such antibody conjugates may be used in the same manner as other conjugated delivery systems where an antibody or other targeting vehicle is conjugated to the biological molecule of interest to provide delivery to desired cells in the in vivo or in vitro environment.
The anti-DNA antibody fragment 3E10 Fv has been demonstrated to be an ideal molecular delivery vehicle due to its efficiency in penetrating into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. Although antibodies that penetrate living cells are frequently toxic or injurious and may explain some of the pathologic manifestations of the autoimmune diseases in which they are found, antibody mAb 3E10, in contrast, shows no harm to cells that it penetrates in tissue culture. Moreover, studies in vitro have shown that mAb 3E10 and scFv fragments of mAb 3E10 can transport relatively large proteins, such as catalase, into the nucleus of cells in tissue culture. Moreover, mAb 3E10 or fragments thereof (e.g., Fv) should not generate significant inflammation in vivo which could hinder therapeutic efficacy of a biologically active molecule conjugated thereto.
It is well known that transporter proteins are involved in the cellular uptake of various molecules into and/or through cells. Carrier-mediated transport systems use proteins that are anchored to the cell membrane, typically by a plurality of membrane-spanning domains and function by transporting their substrates via active or passive mechanisms. Carrier-mediated transport systems are involved in the active or non-active, facilitated transport of many important nutrients such as vitamins, sugars, and amino acids. Carrier-mediated transporters are also present in organs such as the liver and kidney, in which the proteins are involved in the excretion or re-absorption of circulating compounds. Polar or hydrophilic compounds typically diffuse poorly across the lipid bilayers that constitute cellular membranes. For many small molecules (e.g., amino acids, di- and tripeptides, monosaccharides, nucleosides and water-soluble vitamins) there exist specific carrier-mediated transporters for active transport of the solute molecules across biological membranes.
The pathways that allow various molecules to cross cell membranes have been determined. For example, 3E10 Fv is transported by a nucleoside transport pathway allowing it to penetrate cells and localize into the nucleus as discussed in U.S. patent application Ser. No. 12/126,810, filed May 23, 2008; incorporated herein by reference in its entirety.
While various peptides, such as mAb 3E10 Fv have been developed as an intracellular and intranuclear transport vehicle, it has been difficult to produce large amounts of these peptides while maintaining its solubility.